CN115297768A

CN115297768A - Method for determining cognitive disorders

Info

Publication number: CN115297768A
Application number: CN202080091176.6A
Authority: CN
Inventors: J·科温顿
Original assignee: University of Warwick
Current assignee: University of Warwick
Priority date: 2019-10-30
Filing date: 2020-10-29
Publication date: 2022-11-04
Also published as: JP2023500850A; US20220386952A1; WO2021084251A1; EP4051104A1; GB201915753D0

Abstract

A method of determining whether a subject has Mild Cognitive Impairment (MCI) or alzheimer's disease, the method comprising measuring the concentration of one or more VOCs in an exhaled sample from the subject and comparing the concentration to a reference concentration I.

Description

Method for determining cognitive disorders

Technical Field

The present invention relates to a method of determining whether a subject suffers from Mild Cognitive Impairment (MCI). The invention also relates to methods of determining whether a subject has alzheimer's disease. The invention also extends to a method of determining whether a subject has MCI or AD.

Background

The increase in human life inevitably leads to an increase in the prevalence of neurodegenerative diseases (NDDs). Alzheimer's Disease (AD) is a degenerative brain disorder and is the most common cause of dementia. Dementia is characterized by a decline in memory, language, problem solving, planning, reasoning and other basic cognitive abilities that has a direct impact on daily activities. This decline is due to damage or destruction of nerve cells (neurons) in the part of the brain that is involved in cognitive function. While there is currently no cure or method to arrest or slow the progression of dementia, medications, support, and care in the disease can help control symptoms and improve quality of life. In 2017, the global dementia prevalence was estimated to be 5000 million people, with global social costs of 1 trillion dollars due to direct (medical and social care) and indirect (non-paid care for family and friends) costs. It is estimated that its economic impact and cost are higher than those of common chronic diseases such as heart disease and cancer.

The first and mildest phase of AD is the preclinical phase. This stage is followed by Mild Cognitive Impairment (MCI) followed by clinically diagnosable AD. The preclinical phase does not include any significant clinical symptoms; however, there are gradual physiological changes at the cellular level, which are associated with the pathogenesis of the disease. MCI is a transitional stage in the development of AD. Individuals with MCI are defined as individuals with early memory loss and thought problems and are identified based on clinical descriptors rather than diagnosis. Therefore, identifying individuals with MCI requires clinical skill and/or experience. For example, identifying individuals with MCI may include asking for medical history, examining dementia screening blood, performing brain scans (CT, MRI, or PET), and using neuropsychological tests (e.g., using MoCA, ACE-III, and M-ACE sub-scores). It should be noted, however, that MCI is not unique to AD. Thus, not all individuals who develop MCI will develop AD, and some individuals may develop other NDDs, such as Parkinson's Disease (PD) or other medical conditions, such as depression or low vitamin levels. Thus, MCI subjects who are likely to develop AD are typically tested for the presence of biomarkers (e.g., amyloid). For example, if the test result for amyloid is positive, the subject is considered to have MCI due to AD. However, from here on, the term MCI will refer to MCIs that will be possible or will become AD.

After developing AD, an individual may develop dementia, which manifests as impairment in more than one area of cognitive function and impairment in the ability to perform activities of daily living, such as dressing and housework. However, starting from here, the term Alzheimer's Disease (AD) will be used to refer to dementia that progresses from MCI to more than one field of cognitive function and then to impaired activities of daily living.

It is generally accepted that early detection of AD (ideally, in the preclinical stage) will be critical in preventing, slowing, and interrupting the disease. This is when disease modifying treatment is most effective. Some studies have shown that the first brain changes associated with AD may begin more than 20 years before clinical symptoms appear. Identifying biomarkers that can reliably identify the development associated with AD at an early stage would be of high clinical value as it would enable early prophylactic treatment.

Accordingly, there is a need for improved methods of diagnosing and/or differentiating between different stages of alzheimer's disease progression.

Disclosure of Invention

According to a first aspect of the invention, there is provided a method of determining whether a subject has Mild Cognitive Impairment (MCI), the method comprising:

a) Measuring the concentration of one or two VOCs selected from the group consisting of hexanal and heptanal in a sample exhaled by the subject;

b) Comparing the concentration measured in a) with a reference concentration; and

c) Determining that if the concentration measured in a) is more than about 5% higher than the reference concentration, it may indicate that the subject has or will develop MCI.

The inventors have recognized that the difference in VOC concentration in a sample exhaled by a subject from a reference concentration (e.g., a reference concentration in a reference sample from a healthy subject) can be used as a marker to indicate that the subject has or is likely to develop MCI. Thus, the present invention is advantageous because it will enable a clinician to determine early whether a subject has MCI. This in turn will help the clinician to prevent, slow or arrest the progression of the disease, or at least improve the treatment.

Step (a) may further comprise measuring the concentration of one or more other VOCs selected from the group consisting of 2-propanol, acetone, 2-butanone, and 1-butanol in the exhaled sample of the subject.

Determining that the concentration measured in a) is more than about 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% higher or lower than the reference concentration may indicate that the subject has or will develop MCI.

In one embodiment, a subject may be indicated as having or about to develop MCI if the concentration of acetone in the exhaled sample is more than about 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% higher or lower than the reference concentration. Preferably, a higher acetone concentration than the reference concentration indicates that the subject has or will develop MCI.

In one embodiment, a subject may be indicated as having or about to develop MCI if the concentration of 2-butanone in an exhaled sample is more than about 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% higher or lower than a reference concentration. Preferably, a lower concentration of 2-butanone than the reference concentration indicates that the subject has or will develop MCI.

In one embodiment, a subject may be indicated as having or about to develop MCI if the concentration of 2-propanol in an expired sample is more than about 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% higher or lower than a reference concentration. Preferably, a higher concentration of 2-propanol than the reference concentration indicates that the subject has or will develop MCI.

In one embodiment, a subject may be indicated as having or about to develop MCI if the concentration of hexanal in the exhaled sample is more than about 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% higher or lower than the reference concentration. Preferably, a higher hexanal concentration than the reference concentration indicates that the subject has or will develop MCI.

In one embodiment, a subject may be indicated as having or about to develop MCI if the concentration of 1-butanol in an exhaled sample is more than about 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% higher or lower than a reference concentration. Preferably, a 1-butanol concentration higher than the reference concentration indicates that the subject has or will develop MCI.

In one embodiment, a subject may be indicated as having or about to develop MCI if the concentration of heptanal in an exhaled sample is more than about 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% higher or lower than a reference concentration. Preferably, a higher heptanal concentration than the reference concentration indicates that the subject has or will develop MCI.

Preferably, step (a) comprises measuring the concentration of one or more VOCs selected from the group consisting of hexanal, 2-propanol and heptanal in a sample exhaled by the subject. Most preferably, step (a) comprises measuring the concentration of all VOCs in the group consisting of hexanal, 2-propanol and heptanal in the exhaled sample of the subject.

Preferably, determining that the hexanal concentration measured in a) is more than about 100% higher than the reference concentration of hexanal indicates that the subject has or will develop MCI. The method according to the first aspect may further comprise determining that if the hexanal concentration measured in a) is less than about 100% higher than the reference concentration, the subject may be indicated as healthy. The method according to the first aspect may further comprise determining that the subject may be indicated as healthy if the hexanal concentration measured in a) is between about 100% higher and about 100% lower than the reference concentration.

Preferably, determining that the concentration of 2-propanol in a) is more than about 10% higher than the reference concentration of 2-propanol indicates that the subject has or will develop MCI. The method according to the first aspect may further comprise determining that the subject may be indicated as healthy if the concentration of 2-propanol measured in a) is less than about 10% higher than the reference concentration. The method according to the first aspect may further comprise determining that the subject may be indicated as healthy if the concentration of 2-propanol measured in a) is between about 10% higher and about 10% lower than the reference concentration.

Preferably, determining that the concentration of heptanal in a) is more than about 10% greater than the reference concentration of heptanal indicates that the subject has or will develop MCI. The method according to the first aspect may further comprise determining that if the heptanal concentration measured in a) is less than about 10% higher than the reference concentration, the subject may be indicated as healthy. The method according to the first aspect may further comprise determining that the subject may be indicated as healthy if the heptanal concentration measured in a) is between about 10% higher and about 10% lower than the reference concentration.

According to a second aspect, there is provided a method of determining whether a subject has AD, the method comprising:

a) Measuring the concentration of one or two VOCs selected from the group consisting of acetone and 2-butanone in a sample exhaled by the subject;

c) Determining that if the concentration measured in a) is more than about 5% higher or more than about 5% lower than the reference concentration, it may indicate that the subject has or will develop AD.

The inventors have recognized that the difference in VOC concentration in a sample exhaled by a subject from a reference concentration (e.g., a reference concentration from a reference sample of healthy subjects) can be used as a marker to indicate that the subject has or is likely to develop AD. Thus, the present invention is advantageous as it will enable a clinician to determine early whether a subject has AD. This in turn will allow the clinician to prevent, slow or interrupt the progression of the disease, or at least improve the treatment.

Step (a) may further comprise measuring the concentration of one or more other VOCs selected from the group consisting of hexanal, heptanal, 2-propanol and 1-butanol in the sample exhaled by the subject.

Thus, determining that the concentration measured in a) is more than about 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% higher or lower than the reference concentration may indicate that the subject has or will develop AD.

In one embodiment, a subject may be indicated as having or about to develop AD if the concentration of acetone in the exhaled sample is more than about 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% higher or lower than the reference concentration. Preferably, the concentration of acetone in a subject with AD is higher than the reference concentration.

In one embodiment, a subject may be indicated as having or about to develop AD if the concentration of 2-butanone in an exhaled sample is more than about 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% higher or lower than a reference concentration. Preferably, the concentration of 2-butanone in a subject with AD is lower than the reference concentration.

In one embodiment, a subject may be indicated as having or about to develop AD if the concentration of 2-propanol in an expired sample is more than about 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% higher or lower than a reference concentration. Preferably, the concentration of 2-propanol in a subject with AD is higher than a reference concentration.

In one embodiment, a subject may be indicated as having or about to develop AD if the concentration of hexanal in the exhaled sample is more than about 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% higher or lower than the reference concentration. Preferably, the concentration of hexanal in a subject with AD is higher than the reference concentration.

In one embodiment, a subject may be indicated as having or about to develop AD if the concentration of 1-butanol in an exhaled sample is more than about 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% higher or lower than a reference concentration. Preferably, the concentration of 1-butanol in a subject with AD is higher than a reference concentration.

In one embodiment, a subject may be indicated as having or about to develop AD if the concentration of heptanal in an exhaled sample is more than about 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% higher or lower than a reference concentration. Preferably, the concentration of heptanal in a subject with AD is higher than a reference concentration.

Preferably, step (a) comprises measuring the concentration of one or more VOCs selected from the group consisting of acetone, 2-propanol and 2-butanone in a sample exhaled by the subject. Most preferably, step (a) comprises measuring the concentration of all VOCs in the group consisting of acetone, 2-propanol and 2-butanone in the sample exhaled by the subject.

Preferably, the concentration of acetone measured in a) is determined to be higher than the reference concentration of acetone, indicating that the subject has or will develop AD. Most preferably, determining that the acetone concentration measured in a) is more than about 5% higher than the reference concentration of acetone indicates that the subject has or will develop AD. The method according to the second aspect may further comprise determining that the subject may be indicated as healthy if the acetone concentration measured in a) is less than about 5% higher than the reference concentration. The method according to the second aspect may further comprise determining that the subject is indicated as healthy if the acetone concentration measured in a) is between about 5% higher and about 5% lower than the reference concentration.

Preferably, the concentration of 2-propanol measured in a) is determined to be higher than the reference concentration of 2-propanol, indicating that the subject has or will develop AD. Most preferably, determining that the concentration of 2-propanol measured in a) is more than about 10% higher than the reference concentration of 2-propanol indicates that the subject has or will develop AD. The method according to the second aspect may further comprise determining that the subject may be indicated as healthy if the concentration of 2-propanol measured in a) is less than about 10% higher than the reference concentration. The method according to the second aspect may further comprise determining that the subject may be indicated as healthy if the concentration of 2-propanol measured in a) is between about 10% higher and about 10% lower than the reference concentration.

Preferably, the concentration of 2-butanone measured in a) is determined to be lower than the reference concentration of 2-butanone, indicating that the subject suffers from or will develop AD. Most preferably, the concentration of 2-butanone measured in a) is determined to be more than about 5% lower than the reference concentration of 2-butanone, indicating that the subject has or will develop AD. The method according to the second aspect may further comprise determining that the subject may be indicated as healthy if the concentration of 2-butanone measured in a) is less than about 5% below the reference concentration.

According to a third aspect, there is provided a method of determining whether a subject has MCI or AD, the method comprising

a) Measuring the concentration of VOC, 1-butanol in the exhaled sample of the subject;

c) Determining that if the concentration measured in a) is about 5% to about 60% higher than the reference concentration, it is indicative that the subject has or will develop MCI, or

Determining that if the concentration measured in a) is more than about 60% higher than the reference concentration, it may indicate that the subject has or will develop AD.

The inventors have recognized that the difference in VOC concentration between a subject's expired sample and a reference sample (e.g., a reference concentration from a reference sample of a healthy subject) can be used as a marker to distinguish MCI from AD. Thus, VOC concentrations can be used to indicate whether a subject has MCI or AD, or is likely to develop AD. Thus, the present invention will enable clinicians to provide the most appropriate treatment to prevent, slow or interrupt the progression of disease, or at least improve treatment.

Step (a) may further comprise measuring the concentration of one or more other VOCs selected from the group consisting of hexanal, heptanal, 2-propanol, acetone and 2-butanone in the exhaled sample of the subject.

In one embodiment, a subject is indicated as having or about to develop AD if the concentration of acetone in the exhaled sample of the subject is about 5% to about 40% higher, about 5% to about 35% higher, about 5% to about 30% higher, about 5% to about 25% higher, or about 5% to about 20% higher than the reference concentration. A subject is indicated as having or about to develop AD if the concentration of acetone in the sample exhaled by the subject is from about 2.5% to about 40% higher, from about 5% to about 35% higher, from about 10% to about 35% higher, from about 15% to about 35% higher, or from about 20% to about 35% higher than the reference concentration. Thus, if the concentration of acetone in the sample exhaled by the subject is more than about 40%, 35%, 30%, 25%, or 20% higher than the reference concentration, the subject may be indicated as having or about to develop MCI.

In one embodiment, a subject is indicated as having or about to develop AD if the concentration of 2-butanone in an expired sample from the subject is about 2.5% to about 12% lower, or about 5% to about 12% lower than a reference concentration. Thus, if the concentration of 2-butanone in the sample exhaled by the subject is more than about 12% lower than the reference concentration, the subject may be indicated as having or about to develop MCI.

In one embodiment, a subject may be indicated as having or about to develop MCI if the concentration of 2-propanol in the exhaled sample of the subject is about 5% to about 49% higher, about 5% to about 45% higher, about 5% to about 40% higher, about 5% to about 35% higher, about 5% to about 30% higher, or about 5% to about 25% higher than the reference concentration. A subject may be indicated as having or about to develop MCI if the concentration of 2-propanol in the exhaled sample of the subject is about 2.5% to about 49% higher, about 5% to about 45% higher, about 10% to about 45% higher, about 15% to about 45% higher, about 20% to about 45% higher, or about 25% to about 45% higher than the reference concentration. Thus, if the concentration of 2-propanol in the sample exhaled by the subject is more than about 49%, 45%, 40%, 35%, 30% or 25% higher than the reference concentration, the subject may be indicated as having or about to develop AD.

In one embodiment, a subject may be indicated as having or about to develop AD if the concentration of hexanal in the exhaled sample of the subject is about 5% to about 170% higher, about 5% to about 160% higher, about 5% to about 150% higher, about 5% to about 140% higher, about 5% to about 130% higher, about 5% to about 120% higher, about 5% to about 110% higher, about 5% to about 100% higher, about 5% to about 90% higher, about 5% to about 80% higher, about 5% to about 70% higher, about 5% to about 60% higher, or about 5% to about 50% higher than a reference concentration. A subject may be indicated as having or about to develop AD if the concentration of hexanal in a sample exhaled by the subject is about 2.5% to about 170% higher, about 5% to about 160% higher, about 10% to about 150% higher, about 15% to about 140% higher, about 20% to about 130% higher, about 25% to about 130% higher, about 30% to about 130% higher, about 35% to about 130% higher, about 40% to about 130% higher, about 45% to about 130% higher, about 50% to about 130% higher, or about 55% to about 130% higher than a reference concentration. Thus, a subject may be indicated as having or about to develop MCI if the concentration of hexanal in a sample exhaled by the subject is more than about 170%, 160%, 150%, 140%, 130%, 120%, 110%, 100%, 90%, 80%, 70%, 60% or 50% higher than a reference concentration.

In one embodiment, a subject may be indicated as having or about to develop MCI if the concentration of 1-butanol in the exhaled sample of the subject is about 5% to about 80% greater, about 5% to about 75% greater, about 5% to about 70% greater, about 5% to about 65% greater, about 5% to about 60% greater, about 5% to about 55% greater, about 5% to about 50% greater, about 5% to about 45% greater, or about 5% to about 40% greater than the reference concentration. A subject may be indicated as having or about to develop MCI if the concentration of 1-butanol in the exhaled sample of the subject is about 2.5% to about 80% higher, about 5% to about 75% higher, about 10% to about 70% higher, about 15% to about 65% higher, about 20% to about 65% higher, about 25% to about 65% higher, about 30% to about 65% higher, about 35% to about 65% higher, or about 40% to about 65% higher than the reference concentration. Thus, a subject may be indicated as having or about to develop AD if the concentration of 1-butanol in the sample exhaled by the subject is more than about 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, or 40% higher than the reference concentration.

In one embodiment, a subject may be indicated as having or about to develop AD if the concentration of heptanal in the exhaled sample of the subject is about 5% to about 30% greater, about 5% to about 28% greater, about 5% to about 25% greater, about 5% to about 20% greater, or about 5% to about 15% greater than the reference concentration. A subject may be indicated as having or about to develop AD if the concentration of heptanal in the sample exhaled by the subject is about 2.5% to about 30% greater, about 5% to about 28% greater, about 10% to about 25% greater, or about 15% to about 25% greater than the reference concentration. Thus, if the concentration of heptanal in the sample exhaled by the subject is more than about 30%, 28%, 25%, 20%, or 15% higher than the reference concentration, the subject may be indicated as having or about to develop MCI.

The method according to the third aspect may further comprise determining that the subject may be indicated as healthy if the concentration of 1-butanol measured in a) is less than about 5% higher than the reference concentration. The method according to the third aspect may further comprise determining that if the concentration of 1-butanol measured in a) is between about 5% higher and about 5% lower than the reference concentration, the subject may be indicated as healthy.

Preferably, step (a) comprises measuring the concentration of 1-butanol and the concentration of one or both VOCs selected from the group consisting of hexanal and 2-propanol in the sample exhaled by the subject. Most preferably, step (a) comprises measuring the concentration of all VOCs in the group consisting of 1-butanol, hexanal and 2-propanol in the exhaled sample of the subject.

Preferably, the concentration of hexanal measured in a) is determined to be about 5% to about 100% higher than the reference concentration of hexanal, indicating that the subject has or will develop AD. Preferably, it is determined that if the concentration of hexanal measured in a) is more than about 100% higher than the reference concentration, it is indicative that the subject has or will develop MCI. The method according to the third aspect may further comprise determining that if the concentration of hexanal measured in a) is less than about 5% higher than the reference concentration, then the subject is indicated as healthy. The method according to the third aspect may further comprise determining that if the concentration of hexanal measured in a) is between about 5% higher and about 5% lower than the reference concentration, this is indicative that the subject is healthy.

Preferably, the concentration of 2-propanol determined in a) is about 5% to about 49% higher than the reference concentration of 2-propanol, indicating that the subject has or will develop MCI. Preferably, it is determined that if the concentration of 2-propanol measured in a) is more than about 49% higher than the reference concentration, it is indicative that the subject suffers from or will develop AD. The method according to the third aspect may further comprise determining that the concentration of 2-propanol measured in a) is less than about 5% higher than the reference concentration, indicating that the subject is healthy. The method according to the third aspect may further comprise determining that the subject may be indicated as healthy if the concentration of 2-propanol measured in a) is between about 5% higher and about 5% lower than the reference concentration.

The skilled person will understand that determining that if the VOC concentration measured in a) is about the same as the reference concentration indicates that the subject is healthy.

Volatile organic compounds may be defined as organic compounds having a low boiling point at atmospheric pressure. Thus, they form a liquid phase, which evaporates/diffuses to form a gas phase.

As shown in the examples, the inventors surprisingly found (3000 + VOC is present in the breath of a mammal such as a human), that the concentration of a particular VOC present in the exhaled (gas) sample is indicative of whether the subject is healthy, suffering from MCI or AD. Thus, clinicians may use the invention to ensure that patients with AD are given the most appropriate treatment (e.g., palliative and/or pharmacotherapy for symptoms). The age of onset and the rate of progression of AD vary from person to person. In view of this, the present invention is particularly important in identifying individuals who will rapidly develop or are rapidly developing AD.

Furthermore, the present invention is non-invasive and can be readily used to distinguish between healthy individuals, individuals with MCI and individuals with AD within minutes of sampling. Thus, the present invention is a significant improvement over known methods for diagnosing AD, including, for example, testing CSF or performing PET scans. Testing CSF involves an invasive procedure, requiring patient compliance, however PET scanning is complex and expensive.

Thus, the inventors have recognized that the difference in VOC concentrations of a subject's expired sample and a reference sample (e.g., a reference sample from a healthy subject) can be used as a marker suggesting the presence or absence of MCI or AD or that the subject is likely or unlikely to develop MCI or AD.

The skilled person will understand that determining that if the VOC concentration measured in a) is about the same as the reference concentration indicates that the subject is healthy. Thus, for example, a method according to the present invention may further comprise determining that if the concentration of one or more VOCs in a) is between about 5% higher and about 5% lower than the reference concentration, then the subject is indicated as healthy.

It is understood that the detection of a single VOC can be used to identify healthy subjects, subjects with MCI, or subjects with AD. However, the detection of two or more related VOCs can provide a more reliable diagnosis. Hexanal, 2-propanol and heptanal may provide the most reliable diagnosis when differentiating MCI from healthy individuals. Acetone, 2-propanol and 2-butanone may provide the most reliable diagnosis when differentiating between AD and healthy individuals. Hexanal, 2-propanol, and 1-butanol may provide the most reliable diagnoses when distinguishing MCI from AD. The method according to the first, second or third aspect of the invention may be supplemented by other (known) methods.

It is to be understood that the presence or absence and/or concentration of VOCs may be determined using any suitable method/technique/process known in the art, such as gas chromatography-ion mobility spectrometry (GC-IMS) techniques, gas Chromatography (GC), gas chromatography-mass spectrometry (GCMS), mass Spectrometry (MS), ion Mobility Spectrometry (IMS), differential Mobility Spectrometry (DMS), light absorption spectroscopy, field Asymmetric Ion Mobility Spectrometry (FAIMS), electron nose, selective ion flow tube mass spectrometry (SIFT-MS), protein transfer reaction-MS, light absorption/non-dispersive infrared and gas sensors (single or array). Preferably, the step of measuring the concentration of VOCs in the sample exhaled by the subject comprises detecting the concentration of VOCs in the sample by using, for example, GC-IMS techniques.

The reference concentration (for distinguishing between healthy subjects and subjects with MCI, or for distinguishing between healthy subjects and subjects with AD) may be based on a reference sample taken from a healthy subject. The reference concentration may have been obtained by analyzing a statistically significant number of healthy subjects (e.g., 25 or 50 subjects). Thus, the reference concentration may be an average concentration, such as an average, median or mode. Preferably, the average is an average.

Those skilled in the art will appreciate that in embodiments where the presence or absence and/or concentration of a VOC is determined by GC-IMS, the concentration of the VOC may be equivalent to the intensity of the ionic current signal (reference ionic strength) of the VOC.

The reference intensity may be measured in volts. The reference intensity of acetone in a healthy subject may be about 2.66V or in the range of about 2.5V to about 2.9V or about 2.6V to about 2.7V. The reference intensity of 2-butanone in healthy subjects may be about 2.33V or in the range of about 2.2V to about 2.5V or about 2.3V to about 2.5V. The reference intensity of 2-propanol in a healthy subject may be about 1.03V or in the range of about 0.5V to about 1.5V or about 0.75V to about 1.25V. The reference intensity of hexanal in a healthy subject may be about 0.27V or in the range of about 0.1V to about 0.5V or about 0.2V to about 0.4V. The reference intensity of 1-butanol in a healthy subject may be about 0.33V or in the range of about 0.1V to about 0.5V or about 0.2V to about 0.4V. The reference intensity of heptanal in a healthy subject can be about 0.06V or in the range of about 0.01V to about 0.2V or about 0.03V to about 0.1V.

The method according to the present invention may further comprise determining whether the subject has confounding factors that affect the concentration of one or more VOCs in the sample exhaled by the subject. The confounding factors may be selected from the group consisting of 14 units or more of alcohol consumed weekly, over 75 years of age, smoker, and male. The presence of one or more confounders may bias the method according to the invention towards indicating that a subject suffers from or will develop MCI or AD. Thus, preferably, the reference concentration is taken from healthy subjects matched in the confounding selection (who drink 14 units or more weekly, over 75 years of age, smoking and male).

A "subject" can be a human suspected of having AD or MCI. The "subject" can be a vertebrate, mammal, or domestic mammal. Thus, the method according to the invention can be used for diagnosing or treating any animal, such as pigs, cats, dogs, horses, sheep or cattle. Preferably, the subject is a human. The subject may be male or female. The term "healthy" may refer to individuals who do not suffer from AD or MCI. Preferably, a "healthy subject" is a subject without any known pain.

The term "sample" may refer to a specimen taken from the body of a subject. The sample may be an exhalation sample from the nose and/or mouth. Preferably, the sample is an exhaled gas sample.

The term "VOC concentration" may refer to the concentration of one or more VOCs selected from the group consisting of hexanal, 2-propanol, heptanal, acetone, 2-butanone, and 1-butanol.

Drawings

For a better understanding of the present invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying drawings, in which:

FIG. 1 is a 3D topological graph generated by GC-IMS analysis of the composition of an exhaled breath sample from a healthy individual; and

figure 2 is an AUC ROC curve based on analysis of samples taken from different groups of subjects: healthy group vs MCI (blue); healthy group vs AD (black); MCI vs AD (red). Samples were analyzed using a g.a.s.gc-IMS instrument.

Examples

The inventors demonstrate that Volatile Organic Compounds (VOCs) exhaled in the breath can be used as non-invasive biomarkers to distinguish healthy controls from MCI, healthy controls from AD, and MCI from AD. Detection of VOCs is performed using gas chromatography-ion mobility spectrometry (GC-IMS) techniques. Confounding factors such as age, smoking habit, gender and alcohol consumption were also investigated to demonstrate the validity of the results.

Materials and methods

Test subject

A total of 100 subjects were enrolled in the control study. Ethical approval was obtained from the local research ethics committee (reference number 17/18-829, university of princes, uk). MCI and AD patients were treated with Re: cognitive health (princes, uk) was recruited with their respective partners as healthy controls. Volunteers and patients received the information form and indicated consent after the doctor's face-to-face interview. The study cohort included 50 patients (25 MCI,25 AD) and 50 healthy control volunteers. Recruitment of MCI patients was based on an assessment by researchers (inventor s.p.) who reviewed their clinical history and confirmed that subjects exhibited amnesic symptoms that were highly suggestive of AD. Thus, MCI subjects are identified by clinical testing of mental function, complying with current practice as defined by the niaa (National Institute of Aging-Alzheimer's disease association), which defines National standards. Similar clinical criteria apply to the AD group. Specifically, recruitment of AD subjects was based on M-ACE scores below 23, clinical assessment of amnestic problems, and another area of cognitive problems, including dementia patients with dysfunction. Healthy controls include subjects with no known history of neurological disorders (self-report). The mean age of the MCI and AD groups was 74.9 years (standard deviation: 7.6), including 29 males and 21 females. A summary of demographic data for MCI, AD and control subjects, see table 1.

TABLE 1 demographic data of healthy controls and MCI and AD patients

Breath analysis platform

GC-IMS technology is used to detect VOCs in the exhaled breath of a subject. In recent years, portable GC-IMS analyzers, which have been shown to function in medical diagnostics, have been increasing. The BreathSpec used (g.a.s., det, germany) is a commercial instrument consisting of a Gas Chromatograph (GC) and an Ion Mobility Spectrometer (IMS). Importantly, the device (unit) performs the analysis at the point of care, requiring only a few minutes, and is patient friendly, the collected breath sample does not cause stress on the subject. BreathSpec was equipped with an MXT-200 medium polarity column (Thames Restek, studiton, UK) for gas chromatographic separation based on chemical interaction with the column.

Thus, chemicals, including VOCs, in exhaled breath samples are pre-separated by the GC column according to their interaction with the retentate layer within the column. Thus, the chemicals, including VOCs, elute from the column at different times, referred to as residence times. Subsequently, the separated chemical substance (analyte) is further separated by the IMS. The analyte is ionized using a radioactive source and injected into the drift tube using a shutter grid. Under the influence of a uniform electric field (400V/cm), the ions drift against the buffer gas, wherein the various ions acquire different velocities, which are inversely proportional to their size, mass and charge. The ions are then collected on a faraday plate, producing a time-dependent signal corresponding to the ion mobility. The time required for the ions to reach the faraday is referred to as the drift time. This technique can measure substances in the parts per billion (ppb) range and provide measurements in less than 10 minutes. The device recirculates and filters ambient air, which makes the device operable without an external gas supply.

Breath sampling

The sampling process required only 4 seconds of expiratory time. The subject is provided with a disposable plastic mouthpiece that is pushed into the mouthpiece holder/sample inlet and connected directly to the front panel of the instrument. The mouthpiece is open, which allows air within the mouthpiece to escape as exhalation progresses. Thus, the sampling system can separate out the last portion of the exhaled gas, known as the end-tidal or alveolar breath. Alveolar respiration refers to the last part of the exhaled air (350 mL), which is expelled from the lungs and lower respiratory tract, and which is in gas exchange with the blood in the alveoli. The user does not need to exhale until their lungs are emptied as much as possible, but simply requires normal breathing. This improves reproducibility and makes the device suitable for use in vulnerable subjects, such as the elderly.

Typical GC-IMS output responses to breath samples (healthy control subjects in this study) are shown in fig. 1. The samples obtained are represented in a 3D topological graph, where each point is characterized by the retention time (in seconds), drift time (in milliseconds) and ion current signal intensity (in millivolts) in the column. The signal intensity is represented by a color, where each high intensity region represents a chemical or combination of chemicals (having the same characteristics). The long red line is the RIP (active ion peak), which is the background signal. Laboratory analytical explorer (LAV) software (v2.2.1, g.a.s., dtmond, germany) was used for GC-IMS signal observation.

Data analysis

The data analysis methods used were focused on distinguishing three diagnostic groups: AD. MCI and control. However, since the subjects of the control group are more numerous than the MCI subjects or AD subjects, the effect of the unbalanced data set needs to be considered. For this purpose, 25 healthy subjects were randomly selected for analysis. The first step of data analysis includes a pre-processing stage. The purpose of this is to reduce dimensionality, leaving non-background data. A typical GC-IMS data set contains 1100 ten thousand data points, which has a high dimensionality but a low information content. Thus, the target area is cut out from the center of the data set, and then a threshold is applied to remove the background. These steps reduce the number of data points by a factor of 100. Subsequently, a supervised feature selection process was performed, performing class prediction using a k-fold cross-validation method (k =10 in our case). This method involves dividing the original data set into 10 equally sized subsets. Of these 10 subsets, one subset was kept as verification data for the test model, and the remaining 9 subsets were used as training data. Training features were identified using Wilcoxon rank-sum test between the two groups, and those feature points with the lowest p-values were selected and used to construct a model based on five different classifiers. At this stage, these features are identified purely on a statistical basis and not on any biological function. The analysis was run using R (version 3.6.0) and a standard machine learning sub-packet: support Vector Machine (SVM) -kernlab; sparse Logistic Regression (SLR) -glmnet; gaussian process-gbm, neural network-neural net and Random Forest (RF) -random forest. This process was repeated 10 times (number of folds), each subset being used once as validation data. Then, the 10 results are combined to obtain an estimated value, and statistical parameters are calculated therefrom.

In addition to classification analysis, unknown VOCs that are significantly correlated with the efficacy of the generated results can also be identified. Using GC-IMS library search software (v1.0.1, g.a.s., dutmond, germany), we can identify compounds from gas chromatography retention time and ion mobility drift time by referencing the NIST database with approximately 83,000 compound entries. Here, the identified features are plotted back to the original GC-IMS output, and then the chemical species are identified. For quality control, the instrument was normalized using a standard mixture of ketones (2-butanone, 2-pentanone, 2-hexanone, 2-heptanone, 2-octanone, and 2-nonanone) to match the GC-IMS library search software to the equipped column.

Confounding factors

The application of exhaled gas analysis for diagnostic and/or monitoring purposes should take into account possible confounders. These are factors known to have some effect on respiratory content and may therefore introduce bias or create spurious correlations. For example, age is a key confounding factor in this study, as an increase in age is the greatest risk factor for AD. In addition to age, smoking habits, gender and alcohol consumption are also contemplated. After reorganization of patients and volunteers according to confounding factors, the impact of these factors can be evaluated by rerunning the classification analysis applied to the diagnostic group. To simplify the analysis and create a more balanced group, a promiscuous group is defined as: age [ >75 years old vs < =74 years old ], smoking habits [ before never smoking vs/current smoking ], gender [ male vs female ] and drinking [ > =14vsrestricted 14 units/week ]. The latter threshold represents the british guidelines for regular drinking. Table 2 summarizes the confounding factor groups.

Table 2-summary of confounding factor groups.

Results

Example 1 chemical identification

VOC analysis indicates that the concentrations of the three compounds, acetone, 2-propanol and 2-butanone play a crucial role in differentiating healthy controls from AD subjects. The concentrations of 2-propanol, hexanal and 1-butanol varied significantly in the AD vs MCI test. The separation of healthy controls and MCI was dependent on changes in the concentrations of 2-propanol, hexanal and heptanal.

The observed changes measured by GC-IMS for all 100 subjects are shown in table 3.

Example 2 statistical analysis

The analysis results are shown in fig. 2 as overlapping subject operator characteristic (ROC) curves. The corresponding area under the curve (AUC) is a measure of how a parameter can distinguish between the diagnostic (i.e. MCI, AD) and healthy groups. Different comparisons of NPV (negative predictive value) and PPV (positive predictive value) were also calculated. The results of the analysis of the diagnostic panel were obtained using SLR, as shown in table 4.

TABLE 4 GC-IMS diagnostic panel results

Example 3 confounding factors

The previous analyses performed on AD, MCI and healthy groups were repeated for the confounding groups of age, smoking habits, gender and alcohol consumption using the same analysis techniques and algorithms. The analytical results are summarized in table 5.

TABLE 5 GC-IMS confounding factor results

Table 5 shows that possible confounders of gender, smoking and age have no significant effect on breath content (breath content). However, drinking appeared to have the greatest effect on respiration with an AUC of 0.60.

Discussion of the related Art

Although the exact mechanisms associated with the pathogenesis of AD are not fully understood, there is some evidence that defects in mitochondrial metabolism (i.e., alterations in mitochondrial function and potential dysfunction) play a key role in neurodegeneration. In addition, mitochondrial dysfunction in NDD is thought to be associated with increased production of Reactive Oxygen Species (ROS) that cause cell damage and intercellular oxidative stress. Endogenous VOCs (produced in vivo) follow metabolic pathways, are transported to the lungs through the bloodstream, and are exhaled in the pulmonary breath. Oxidative stress has been detected in the blood, thus providing an opportunity to apply respiration analysis to facilitate the discovery and assessment of biomarkers associated with cellular energy metabolism, mitochondrial dysfunction and oxidative stress.

A key advantage of the results in this application is that the age and gender of the subject population are approximately balanced. Furthermore, by recruiting MCI and AD patients and their respective partners as healthy controls, it is possible to design more reliable experiments and minimize the possible impact of lifestyle and environmental factors that may introduce significant interpersonal variability. In addition, age matching is a key factor to consider because more women are suffering from AD and other forms of dementia than men. For example, almost two thirds of americans with alzheimer's disease are women. It is believed that this difference is due to women generally having a longer life than men, which increases the risk factors for developing AD. However, there are other factors, such as sex-specific genetics and hormonal factors, which can lead to differences in clinical efficacy. Furthermore, depending on gender, lifestyle choices such as smoking, excessive drinking, poor diet and the resulting health conditions (obesity, type 2 diabetes and cardiovascular disease) can have different effects on the risk of dementia. In this analysis, the age is subdivided into "young elderly" (65-74 years) and "older elderly" (75 years or older). This division explains the dramatic rise in Alzheimer's disease cases above 75 years- -15% of patients with Alzheimer's disease are between 65 and 75 years of age, and 44% are between 75 and 85 years of age.

VOC analysis showed that acetone, 2-propanol and 2-butanone significantly contributed to our efficacy analysis of AD compared to the control group. These compounds are often associated with normal breathing. Similarly, changes in other respiratory markers, such as 2-propanol, hexanal, heptanal, and 1-butanol, facilitated the separation of AD vs MCI and MCI vs control experiments. This indicates that AD-related respiratory changes have subtle effects on the overall respiratory content. The change in acetone is of particular interest. The early stages of AD are associated with a regiospecific decline in brain glucose metabolism. This can be supplemented by ketone bodies including acetoacetate, β -hydroxybutyrate and acetone. These are usually produced from fat stores when glucose is not available (e.g. during prolonged fasting or when subscribing to a ketogenic diet), which may lead to an increase in acetone levels in the exhaled breath of AD patients. AD patients often have poor appetite, with approximately half of mild-AD subjects reporting altered appetite. Changes in exhaled acetone observed in this study may be related to this phenomenon. To our knowledge, there is currently no known association between 1-butanol and the metabolic pathways associated with AD or other NDDs.

Analysis of possible confounders showed that gender, smoking, age and alcohol consumption had no significant effect on respiratory content. Of these factors, alcohol appears to have the greatest effect, with an AUC of about 0.60. However, this factor is not sufficient to form two distinct groups, nor to disrupt the AD-related analysis.

Conclusion

The results of this study demonstrate the potential utility of analyzing respiratory VOCs in differentiating between MCI, AD, and healthy controls. Although this is a simple study with a relatively clear/well-defined set, the methods used were always able to distinguish between diagnostic sets [ AUC +95%, sensitivity, specificity ], healthy set vs MCI: [0.77 (0.64-0.9), 0.68,0.8], healthy group vs AD: [0.83 (0.72-0.94), 0.6,0.96], and MCI vs AD: [0.70 (0.55-0.85),0.6,0.84]. Analysis of possible confounders showed that gender, age, smoking habits and alcohol consumption had no significant effect on respiratory content. VOC analysis indicates that six compounds, temporarily identified as acetone, 2-propanol, 2-butanone, hexanal, heptanal and 1-butanol, play a crucial role in differentiating diagnostic groups. GC-IMS analysis techniques have proven suitable for non-invasive sampling of elderly subjects and show potential as a rapid, high-throughput, AD real-time diagnostic tool in a point-of-care clinical setting.

Claims

1. A method of determining whether a subject has Mild Cognitive Impairment (MCI), the method comprising:

c) Determining that if the concentration measured in a) is more than about 5% higher than the reference concentration, it is indicative that the subject has or will develop MCI.

2. The method of claim 1, wherein (a) further comprises measuring the concentration of 2-propanol in the sample exhaled by the subject.

3. The method of claim 1 or claim 2, wherein determining that the hexanal concentration measured in a) is more than about 100% higher than the reference concentration of hexanal indicates that the subject has or will develop MCI.

4. The method of claim 2, wherein determining that the concentration of 2-propanol in a) is more than about 10% higher than the reference concentration of 2-propanol indicates that the subject has or will develop MCI.

5. The method of any one of the preceding claims, wherein determining that the concentration of heptanal in a) is more than about 10% greater than the reference concentration of heptanal indicates that the subject has or will develop MCI.

6. A method of determining whether a subject has AD, the method comprising:

b) Measuring the concentration of one or two VOCs selected from the group consisting of acetone and 2-butanone in a sample exhaled by the subject;

c) Determining that if the concentration measured in a) is more than about 5% higher or more than about 5% lower than the reference concentration, it is indicative that the subject suffers from or will develop AD.

7. The method of claim 6, wherein (a) further comprises measuring the concentration of 2-propanol in an exhaled sample of the subject, optionally wherein determining that the concentration of 2-propanol measured in a) is higher than a reference concentration of 2-propanol indicates that the subject has or will develop AD.

8. The method of claim 6 or claim 7, wherein determining that the concentration of acetone measured in a) is higher than the reference concentration of acetone indicates that the subject has or will develop AD, optionally wherein determining that the concentration of acetone measured in a) is more than about 5% higher than the reference concentration of acetone indicates that the subject has or will develop AD.

9. The method of claim 7, wherein determining that the concentration of 2-propanol measured in a) is more than about 10% higher than the reference concentration of 2-propanol indicates that the subject has or will develop AD.

10. The method according to any one of claims 6 to 9, wherein determining that the concentration of 2-butanone measured in a) is lower than the reference concentration of 2-butanone indicates that the subject has or will develop AD.

11. The method of claim 10, wherein determining that the concentration of 2-butanone measured in a) is more than about 5% below the reference concentration of 2-butanone indicates that the subject has or will develop AD.

12. A method of determining whether a subject has MCI or AD, the method comprising

c) Determining that if the concentration measured in a) is about 5% to about 60% higher than the reference concentration, then indicating that the subject has or will develop MCI, or

Determining that if the concentration measured in a) is more than about 60% higher than the reference concentration, it is indicative that the subject has or will develop AD.

13. The method of claim 12, wherein (a) comprises measuring the concentration of 1-butanol and the concentration of one or two VOCs selected from the group consisting of hexanal and 2-propanol in the sample exhaled by the subject.

14. The method of claim 13, wherein determining that the hexanal concentration measured in a) is about 5% to about 100% higher than the reference concentration of hexanal indicates that the subject has or will develop AD.

15. The method of claim 14, wherein determining that the concentration of 2-propanol measured in a) is about 5% to about 49% higher than the reference concentration of 2-propanol indicates that the subject has or will develop MCI.